This invention relates to plant genetic engineering. In particular, the invention is for the production of transformed plants in which only sequences between the right border and left border elements of Agrobacterium are obtained in selected plant cells.
Agrobacterium-mediated DNA delivery is a routine method for delivering DNA to plant cells for the purpose of producing genetically modified plants. Typically, these methods involve the use of disarmed Agrobacterium strains in which genes of interest are incorporated between right border and left border elements of a Ti plasmid. Work done in the late 1970s and early 1980s provided evidence that the sequences between the right border (RB) and left border (LB) were found in plant cells of tumor lines obtained following infection with wild isolates of Agrobacterium tumefaciens (Chilton et al., Nature, 275:147-49 (1978); Depicker et al., Nature, 275:150-53 (1978)). This sequence, commonly transferred to plant tumors, was taken to be the T-DNA, although it was known then that sequences in addition to this xe2x80x9ccommon DNAxe2x80x9d could be found in tumor cells (Thomashow et al., Cell, 19:729-39 (1980)).
The RB sequence has since been shown to function as a site for nucleolytic cleavage by virD2 protein, and initiation of replicative DNA transfer (Albright et al., J. Bacteriol. 169:1046-55 (1987), and Howard et al. DNAs 86:4017-21 (1989)). The LB element, with a sequence very similar to that of the RB, is the site of termination for transfer of sequences between the RB and LB. The LB element has also been shown to be a site for nucleolytic cleavage (Albright et al., J. Bacteriol. 169:1046-55(1987)). Albright et al. proposed that following cleavage at either border sequence, replication is initiated unidirectionally using a strand-displacement mechanism.
Based on this understanding of T-DNA transfer, plant molecular biologists engineered recombinant T-DNAs in which right and left border elements were placed around sequences they desired to transfer to plant cells. These recombinant T-DNA""s have been used successfully for introducing the sequences between right and left border elements into plant cells. Schilperoort and his colleagues (see, e.g., U.S. Pat. No. 5,464,763) demonstrated that artificial T-DNAs could be constructed in a vector distinct from the Ti plasmid itself, which contained the functions necessary for transfer of T-DNA sequences into plant cells. Such T-DNA vectors have been used commonly by plant genetic engineers, in combination with disarmed Agrobacterium strains, for the transfer of engineered genes into plant cells.
Recently, a number of authors have reported the transfer of sequences other than those between right and left border elements into plant cells that have been selected for the presence of sequences that lie between the borders (Kononov et al., Plant J., 11:945-57, (1997); Wenck et al., Plant Mol. Biol., 34:913-22 (1997). A variable number of plant cells selected for transfer of sequences between the border elements also contain sequences beyond the left border. The frequency varies from 5% to over 75% (Martineau et al., Plant Cell, 6: 1032-33 (1994)). These sequences are thought to be transferred to the DNA either as a result of failure to terminate transfer at the left border, or by virtue of transfer initiated at the left border. In some examples of transgenic plants carrying sequences beyond the left border, the non-T-DNA sequences are integrated at locations distinct from ones carrying between border sequences.
It is often desirable to produce a population of transgenic plants that contain only those sequences bounded by the border elements. Although means for identifying such plant have been proposed (see, e.g., Ramanathan and Veluthambi Plant Mol. Biol., 28: 1149-54, 1995), the art lacks simple and cost effective methods for producing a population of transgenic plants in which the number of transgenic individuals with non-T-DNA sequences is reduced substantially. The present invention provides these and other advantages.
The present invention provides methods for eliminating plants containing non-T-DNA sequences derived from a T-DNA vector. More specifically, the present invention provides a method for killing plant cells that receive non-T-DNA sequences derived from the T-DNA vector where the method is based on incorporation of a lethal polynucleotide sequence into the non-T-DNA portion of the vector.
The methods comprise introducing into plant cells a T-DNA vector comprising a T-DNA sequence having a right border, a left border and the polynucleotide of interest positioned between the right border and the left border. Also included in the vector is a non-T-DNA sequence comprising a lethal polynucleotide sequence. Plant cells are then selected which comprise the T-DNA sequence and do not comprise the lethal polynucleotide sequence. The plant cells are then regenerated into transgenic plants.
The lethal polynucleotide can encode a lethal polypeptide (e.g., a ribonuclease) or encode a lethal mRNA transcript (e.g., a ribozyme or antisense RNA). In some embodiments, the ribonuclease is Barnase. The lethal polynucleotide may be altered to prevent expression in the Agrobacterium host. This can be accomplished, for instance, by including an intron in the coding region. The non-T-DNA sequence may further comprise a screenable marker and the method may further comprise detection of the screenable marker in the plant cells. Exemplary screenable markers include xcex2-glucuronidase.
The invention also provides vectors useful in the methods of the invention, as well as plant cells made by the methods.